Immunological adjuvants are used in combination with vaccines to augment the immune response to the antigen. One way in which immunological adjuvants function is by attracting macrophages to the antigen, so that the macrophages can present the antigen to the regional lymph nodes and initiate an effective antigenic response. Adjuvants may also act as carriers themselves for the antigen, or may influence the immune response by other mechanisms such as depot effect, cytokine induction, complement activation, recruiting of different cell populations of the immunological system, antigen delivery to different antigen presenting cells, regulation of the expression of HLA class I or class II molecules and the stimulation to produce different antibody subtypes. Many of the newer vaccines are only weakly immunogenic and thus require the presence of adjuvants.
Materials having adjuvant activity are well known. Alum (Al(OH)3), and similar aluminum gels are adjuvants licensed for human use. The adjuvant activity of alum was first discovered in 1926 by Glenny (Chemistry and Industry, Jun. 15, 1926; J. Path. Bacteriol, 34, 267). Aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum.
Other materials are also known to have adjuvant activity, and these include: Freund's complete adjuvant, a water-in-mineral-oil emulsion which contains killed, dried mycobacteria in the oil phase; Freund's incomplete adjuvant, a weaker formulation without the mycobacteria; saponin, a membrane active glucoside extracted from the tree Quillia saponaria; nonionic block copolymer surfactants, non-metabolised synthetic molecules which tend to bind proteins to cell surfaces; ISCOMS, lipid micelles incorporating Quil A (saponin) which mimic, in physical terms, infectious particles; and muramyl dipeptide, a leukocyte stimulatory molecule that is one of the active components of killed mycobacteria. A known adjuvant in cancer therapy is bacillus calmette guerin (BCG) which is used in combination with various anti-cancer vaccine strategies. GM-CSF has also been found to be an effective adjuvant when used in combination with autologous tumor cells.
With all of these agents, toxicity, unacceptable chronic reactions and/or low potency (in the case of BCG) are features which currently limit their use as potential adjuvants. Thus there is an ongoing and current need for new adjuvants to boost the human immune response to vaccines, in both cancer therapy and other disease treatments.
One line of research in the development of adjuvents has been directed to the study of dendritic cells. Dendritic cells (DC) are professional antigen presenting cells (APC) that have the unique capacity to initiate primary immune responses in vivo and in vitro (1-3). They are derived from myeloid (DC1) or lymphoid (DC2) precursors and are distributed in their immature form throughout the body in tissues that commonly encounter environmental pathogens (skin, mucus membranes, gut epithelia, etc.) (1, 2, 4-7). Whereas DC1 and DC2 comprise a small percentage of the total number of mononuclear cells in the peripheral circulation, DC1 precursors in the form of CD14+/CD11c+/HLA-DR+ monocytes are relatively abundant, constituting about 10% to 15% of mononuclear blood cells (11-15).
Immature DC express a host of surface structures that are involved in antigen acquisition, DC activation/maturation, and antigen presentation (1, 2, 8). Once DC encounter antigen, they undergo a maturation process characterized by the up-regulation of HLA class I and II molecules as well as co-stimulatory molecules and interact with cognate receptors on T and B lymphocytes, resulting in the generation of antigen specific cellular and humoral immune responses (1, 2, 9, 10).
DC are considered to be the primary APC in the immune system. The ability to isolate these cells and/or their precursors and to study them in vitro has added considerable dimension to knowledge of their role in innate and acquired immunity (1, 2). The classic means of generating human DC in vitro is to isolate and enrich CD14+-monocytes from peripheral blood and culture them for various periods of time in GM-CSF and IL-4 followed by final maturation with a number of cytokines, including IL-2, IL-6, IL-7, IL-13, IL-15, TNFα, IL-1β, (16, 36) or with various other agents including lipopolysaccharides, PGE2, type 1 interferons, or double-stranded RNA (20-24).
Numerous investigators have shown that these in vitro generated monocyte-derived DC are potent antigen presenting cells (APC) capable of initiating primary and recall antigen-specific CD4+ and CD8+ T cell responses (27-30). Recent in vitro studies have generated a rather extensive body of information regarding the biology of DC1 and shed light on the processes whereby antigen specific immune responses are generated in vivo (1-2). In the peripheral tissues, immature DC acquire antigenic materials in the context of danger signals initiating a complex cytokine/chemokine milieu that is generated by DC and other cell types in the vicinity (31). Soluble mediators produced by DC may act in an autocrine or paracrine fashion. T cells produce additional cytokines and chemokines following interaction with antigen armed DC, as do other immune cells that are activated by the cytokines released (32-35). This complex network of interactions may in turn create an environment that promotes the generation of DC from their monocyte precursors.
Several investigators have described the use of various cell-free culture supernatants, also referred to as “conditioned media” as DC maturation agents. These media contain more or less well defined mixtures of cytokines (12, 25, 26). Monocyte conditioned media (MCM), containing IFNα, IL-1β, IL-6, and TNFα, has been shown to induce expression of CD83 and p55, surface molecules that are characteristic of mature DC (26). However, when combinations of these cytokines were added to immature DC at concentrations comparable to those found in the conditioned media, they were less effective in maturing DC compared to MCM. These results suggest that additional components were required to affect full maturation of immature DC.
In one study, Kato et al prepared conditioned media (designated TCCM) by culturing isolated T cells with anti-CD3 monoclonal antibodies that had been adhered to plastic surfaces (25). This media was able to mature immature DC that had been generated from monocytes cultured in (GM-CSF and IL-4. Interestingly, different clones of anti-CD3 induced different quantities of soluble CD40 ligand and IFNγ and these differences were reflected in the capacity of the media to mature DC.
Whereas MCM and TCCM are very effective mediators of final DC maturation, their capacity to differentiate monocytes into immature DC was not reported. The inventors are aware of one report where this activity was observed with media from PBMC stimulated with CpG-A oligonucleotides (33). It is well established that CpG-A induces type 1 interferons (IFNα/IFNβ) production by plasmacytoid DCs, a minor cellular component in PBMC (6, 37-39). In the cited study, antibodies to IFNα diminished but did not abrogate the activity of this culture media suggesting that additional cytokines might be participating in the induction of monocyte differentiation. This is certainly possible since IFNα is known to induce production of cytokines in other cell types (including T cells) that may affect monocyte differentiation (6, 38, 39).
It is thought that compounds or compositions which promote that maturation of dendritic cells, when administered in combination with a vaccine antigen, will result in more antigen presenting cells presenting the vaccine antigen to T lymphocytes and B cells, thus bolstering the immune response to the vaccine antigen.